1. Field of the Invention
The present invention is concerned with methods of determining substrate utilization in living tissue. More particularly, the methods employ NMR isotopomer analysis of .sup.13 C-labeled substrate spectra obtained from the tissue, allowing a rapid, single measurement under nonsteady state conditions of substrate utilization which may be used to assess tissue damage. The analysis utilizes a homonuclear decoupling method of spectrum simplification providing collapse of a multiplet to allow precise determination of peak areas.
2. Description of Related Art
The relative rates of utilization of various exogenous and/or endogenous substrates in normal cells and tissues may be sensitive to many factors including cellular work rate, physiologic state, drugs, toxins, hormones, and the like. Substrate utilization may also be sensitive to disease states such as ischemia, infection, inflammation, trauma, congenital defects in metabolism, acquired defects in metabolism, or during malignant transformations such as cancer. Thus, precise quantitation of substrate utilization could have broad application since it may provide insight into the integrated functional state and viability of cells or a tissue.
Acetyl Coenzyme A (acetyl-CoA) is a key intermediate in cellular biochemistry. It is oxidized in the citric acid cycle for the production of energy, and it is a precursor in multiple biosynthetic processes. Acetyl-CoA may be derived frown numerous compounds, each of which must be metabolized through different pathways subject to complex and interacting regulatory processes. Thus, the relative contribution of one or more substrates to acetyl-CoA reflects cellular metabolic state. Since this measurement is so important for the understanding of tissue metabolism, it has been the objective of numerous studies in many cellular preparations and tissues, particularly heart tissue (more recent citations include, for example, Jue, et al., 1987; Avison, et al., 1988; Bottomley, et al., 1989; Heershap, et al., 1990; Shuhnan, et al., 1990; Rothman, et al., 1991; and Magnusson, et al., 1992).
The measurement of the contribution of a compound to acetyl-CoA ordinarily requires an estimate of the rate of acetyl-CoA utilization, typically from oxygen consumption, and the rate of substrate utilization under steady-state conditions. The latter usually is measured by the rate of appearance of .sup.14 CO.sub.2 from a .sup.14 C-enriched substrate, the rate of removal of substrate from the perfusion medium, or multiexponential analysis of .sup.11 C time-activity curves in tissues utilizing .sup.11 C-enriched substrates.
However, substrate and oxygen removal are difficult to measure under some important conditions, and metabolic and isotopic steady-state often cannot be assured. Further, since pyruvate may be metabolized by either pyruvate dehydrogenase or through a pyruvate carboxylation pathway, the appearance of .sup.14 CO.sub.2 from .sup.14 C-enriched pyruvate (or its precursors) indicates net substrate oxidation only if the carbon skeleton enters the citric acid cycle via pyruvate dehydrogenase. Similarly, .sup.14 CO.sub.2 release from fatty acids is an unreliable measure of this oxidation (Chatzidakis and Otto, 1987; Veerkamp, et al., 1986). Thus, standard methods for assessing substrate competition and oxidation are often not satisfactory for rapidly changing or spatially heterogeneous metabolic states, or if more than one pathway is available for carbon flow into the citric acid cycle.
In spite of these limitations on traditional methods, there is substantial interest in the measurement of substrate oxidation for the assessment of tissue metabolism and viability. For example, position emission tomography (PET) has been used to examine regional myocardial metabolism during ischemic and other states. However, the interpretation of some PET observations is controversial, for example, fatty acid oxidation in ischemic reperfused myocardium. PET studies generally have concluded that fatty acid oxidation is suppressed, but other reports have not validated this finding (Mickel, et al., 1986; Myears, et al., 1987: Liedke, et al., 1988; Wyns, et al., 1989: Lerch, et al., 1981; Schwaiger, et al., 1985). PET is fundamentally limited by the lack of knowledge of the chemical state of the tracer. For example, a compound may enter a cell where it may be trapped and stored, metabolized to acetyl-CoA and oxidized, or it may remain in the cell briefly and then diffuse out, unchanged. Numerous assumptions regarding the metabolic fate of a tracer are therefore required.
For these reasons, some recent PET studies have emphasized the utilization of a very simple compound, acetate, which is not subject to many of the complex physiological processes which regulate normal metabolism (Brown, et al., 1987). Analysis of the results is thereby simplified, but acetate is not a physiological substrate. Biochemical and physiological studies using .sup.11 C are also limited by the problem of working with a radioactive element with a very short half-life. Thus, a nearby cyclotron is essential, and rapid chemical synthesis is required. The study of some molecules or certain labeling patterns is simply not practical.
The analogous use of .sup.13 C enriched substrates to monitor intermediary metabolism has been established (London, 1988). Multiple enriched intermediates of the citric acid cycle may be detected by NMR spectroscopy (London, 1988; Walker, et al., 1982; Chance, et al., 1983: Cohen, 1983; Walker and London, 1987; Malloy, et al., 1990A). It has been shown that citric acid cycle flux may be determined if the fractional enrichments in intermediates are measured repeatedly after the addition of enriched substrate (Chance, et al., 1983). This method, however, assumes steady-state flux conditions, constant intermediate pool sizes, and good temporal resolution. Although collection of in vivo data is theoretically possible, the method depends on measurement of fractional enrichment in glutamate and other intermediates, a requirement which may be difficult to meet under many important conditions.
An alternative to the measurement of absolute citric acid cycle flux is the measurement of the relative rates of competing pathways feeding acetyl-CoA. This approach has been reported previously. In some instances, metabolic and isotopic steady-state were assumed for the purposes of data analysis and these conditions were established experimentally (Malloy, et al., 1988, 1990A; Sherry, et al., 1988). Other reports indicated that insight into the pathways feeding acetyl-CoA could be obtained by .sup.13 C NMR spectroscopy (Cohen, 1983; Walker and London, 1987). Finally, recent reports describe how to measure the ratio of the contribution of two labeled substrates to acetyl-CoA under nonsteady-state conditions (Malloy, 1990B, 1990C; Sherry, et al., 1992). Up to three labeled substances can be analyzed and the fraction of unlabeled acetyl CoA can be determined by a non steady state analysis (Malloy, et al., 1990B).
.sup.13 C NMR is useful for the monitoring of metabolism of .sup.13 C labeled compounds in experimental animals and humans. However, there are three important factors limiting study of substrate utilization in vivo. First, there is the consideration of expense. Significant amounts of relatively expensive labeled compounds make it difficult to maintain a constant concentration in the blood for the time required to attain isotopic steady-state. Second, many conditions of interest may involve rapidly changing metabolic conditions, and metabolic state cannot be assumed. Finally, an isotopomer analysis applicable in vivo to determine .sup.13 C contributions to the carbon skeleton of citric acid cycle components has been applied only when B.sub.0 homogeneity was sufficient to allow resolution of .sup.13 C-.sup.13 C scalar coupling. This condition of homogeneity does not occur under the circumstances of many in vivo measurements.
.sup.13 C in labeled compounds can be detected in humans. The most widely examined pathways in humans to date have been those involving either storage of glucose or mobilization of glycogen. The C 1 resonance of glycogen has been detected by numerous groups in both human liver and muscle. The rate of net hepatic glycogenolysis in fasted humans has been determined by monitoring the time dependent signal of natural abundance glycogen. This in combination with glucose production rates measured by turnover of [6-.sup.3 H]glucose, has allowed an estimate of gluconeogenic rates in normal controls and in patients with type II diabetes mellitus. [1-.sup.13 C]glucose levels in human brain have also been quantitated to yield an estimate of glucose transport rates. Some metabolic end-products of [1-.sup.13 C]glucose have also been detected in human brain and incorporation of .sup.13 C into [4-.sup.13 C]glutamate has been used to estimate citric acid cycle flux.
Thus .sup.13 C from labeled isotopes can be detected in vivo and isotopomer methods provide substantial information. Although the isotopomer method would be of most practical value for in vivo applications, it has until now not yet been successfully applied, largely because of difficulties in resolving those multiplets in spectra obtained from intact tissue. There is thus a need to develop methods for quantitative substrate selection in intact tissue in the whole, live animal. This would be of particular benefit in recent heart attack patients to determine damaging effects of ischemia. Similarly, analyses of tissue damage for stroke and brain injured patients could be obtained rapidly and efficiently with a single measurement.